1. Field of the Invention
The present invention relates to scanning systems for examining biological material and, in particular, to pixel clocking in optical scanning systems having a laser to excite fluorescently tagged biological materials.
2. Related Art
Synthesized nucleic acid probe arrays, such as Affymetrix(copyright) GeneChip(copyright) synthesized probe arrays, have been used to generate unprecedented amounts of information about biological systems. For example, a commercially available GeneChip (copyright) array set from Affymetrix, Inc. of Santa Clara, Calif., is capable of monitoring the expression levels of approximately 6,500 murine genes and expressed sequence tags (EST""s). Experimenters can quickly design follow-on experiments with respect to genes, EST""s, or other biological materials of interest by, for example, producing in their own laboratories microscope slides containing dense arrays of probes using the Affymetrix(copyright) 417(trademark) or 427(trademark) Arrayers or other spotting devices. Analysis of data from experiments with synthesized and/or spotted probe arrays may lead to the development of new drugs and new diagnostic tools.
In some conventional applications, this analysis begins with the capture of fluorescent signals indicating hybridization of labeled target samples with probes on synthesized or spotted probe arrays. The devices used to capture these signals often are referred to as scanners. Due to the relatively small emission signals sometimes available from the hybridized target-probe pairs, the presence of background fluorescent signals, the high density of the arrays, variations in the responsiveness of various fluorescent labels, and other factors, care must be taken in designing scanners to properly acquire and process the fluorescent signals indicating hybridization. For example, U.S. Pat. No. 6,171,793 to Phillips, et al., hereby incorporated herein in its entirety for all purposes, describes a method for scanning probe arrays to provide data having a dynamic range that exceeds that of the scanner. As another example, U.S. patent application Ser. No. 09/681,819, filed Jun. 11, 2001 and hereby incorporated herein by reference in its entirety for all purposes, describes systems and methods for aligning grids on scanned images to provide appropriate pixel analysis. Nonetheless, there is a continuing need to improve scanner design to provide more accurate and reliable fluorescent signals and thus provide experimenters with more sensitive and accurate data.
High-density probe arrays allow higher throughput and other advantages as compared to probe arrays of lower densities. However, as the density of probes grows, the difficulties also generally increase of accurately and reliably distinguishing adjacent probes and collecting sufficient image information, i.e., pixels, specific to each probe. Similar difficulties emerge as the goal of higher throughput results in faster image scans. There thus is a need for methods and systems for scanning high-density probe arrays, possibly at higher scanning speeds, but yet preserving high sensitivity, accuracy, and reliability with respect to the acquisition of image pixels.
Systems, methods, and products to address these and other needs are described herein with respect to illustrative, non-limiting, implementations. Various alternatives, modifications and equivalents are possible. For example, certain systems, methods, and computer software products are described herein using exemplary implementations for analyzing data from arrays of biological materials produced by the Affymetrix(copyright) 417(trademark) or 427(trademark) Arrayer. Other illustrative implementations are referred to in relation to data from Affymetrix(copyright) GeneChip(copyright) probe arrays. However, these systems, methods, and products may be applied with respect to many other types of probe arrays and, more generally, with respect to numerous parallel biological assays produced in accordance with other conventional technologies and/or produced in accordance with techniques that may be developed in the future. For example, the systems, methods, and products described herein may be applied to parallel assays of nucleic acids, PCR products generated from cDNA clones, proteins, antibodies, or many other biological materials. These materials may be disposed on slides (as typically used for spotted arrays), on substrates employed for GeneChip arrays, or on beads, optical fibers, or other substrates or media. Moreover, the probes need not be immobilized in or on a substrate, and, if immobilized, need not be disposed in regular patterns or arrays. For convenience, the term probe array will generally be used broadly hereafter to refer to all of these types of arrays and parallel biological assays.
In accordance with one preferred embodiment, a method is described that includes the steps of (1) directing an excitation beam to a plurality of probe locations, each probe location including one or more probe molecules; (2) receiving an emission signal responsive to the excitation beam exciting one or more probe locations; (3) determining a plurality of radial positions of the excitation beam; (4) comparing the plurality of radial positions to a plurality of position data; (5) generating a clock signal based, at least in part, on comparing one or more of the plurality of radial positions with one or more of the plurality of position data; and (6) analyzing molecules at one or more probe locations based, at least in part, on one or more values of the emission signal determined by the clock signal.
In accordance with another preferred embodiment, an apparatus is described that includes an emission receiving element that receives an emission signal from a plurality of locations of a probe array. The emission receiving element could be, for example, an objective lens that may be disposed at the end of a scanning arm. The apparatus also includes a radial position generator that generates a plurality of radial positions of the emission receiving element. The radial position generator could be, as a non-limiting example, a position transducer associated with a galvanometer that moves the scanning arm in arcs. Another element of the apparatus of this embodiment is a memory unit having stored therein a plurality of position data, each representing a radial position of the emission receiving element. The memory unit could, for example, be included in the scanner or in a computer coupled to the scanner either locally or remotely. Yet another element of this apparatus is a comparator that generates a clock signal based, at least in part, on comparing one or more of the plurality of radial positions with one or more of the plurality of position data. The clock signal may include digital clock pulses. The comparison could be accomplished, as one example, by converting analog radial position signals to digital values and comparing them with a table of position data in a database or look-up table. In some implementations, the apparatus also includes a sampler that samples the emission signal when enabled by the clock signal. Other elements that may be included are an excitation-beam-delivering element that deliver an excitation beam to the plurality of locations of the probe array, and/or a scanning member that scans the probe array. The emission receiving element may be moved with respect to the probe array by the scanning member that may be, as noted, a scanning arm. A moving member, such as a galvanometer for example, may also be included for moving the scanning arm. The moving member may move the scanning arm in bi-directional arcs in a plane. In some implementations, the plurality of position data are determined so that a first linear distance between a first position of the emission receiving element corresponding to a first radial position and a second position of the emission receiving element corresponding to a second radial position is equal to a second linear distance between the second position of the emission receiving element and a third position of the emission receiving element corresponding to a third radial position. As one example, the position data may be predetermined so that they correspond to radial positions of the objective lens as it scans in arcs over a substrate of a probe array such that those radial positions correspond to equally spaced points on a straight line across the substrate. The probe array may, as non-limiting examples, be a synthesized or spotted probe array.
A method is also described in accordance with yet other preferred embodiments that includes the steps of: (1) detecting a signal from a plurality of locations of a probe array; (2) generating a plurality of radial positions corresponding with two or more of the plurality of locations of the probe array; and (3) comparing one or more of the plurality of radial positions with one or more of a plurality of position data. Other steps may include (4) generating a clock signal responsive to step (3); and (5) sampling the signal when enabled by the clock signal. Step (1) may include scanning the probe array in arcs, which may be bi-directional. Also, step (1) in some implementations may include scanning the probe array in a first arcuate scan line two or more times before scanning the probe array in a second arcuate scan line.
In accordance with a further preferred embodiment, an apparatus is described that includes an emission receiving element that receives an emission signal from a plurality of locations of a probe array, and a clock signal generator that generates a clock signal based, at least in part, on radial position data. The apparatus may also have a sampler that samples the emission signal when enabled by the clock signal. In other embodiments, a method is described that includes the steps of receiving an emission signal from a plurality of locations of a probe array, and generating a clock signal based, at least in part, on radial position data. In accordance with yet other embodiments, a position-based pixel clock sampling apparatus is described for enabling the sampling of an emission signal detected using a scanner having a scanning arm. The apparatus includes a transducer that provides an analog position signal representing a plurality of radial positions of the scanning arm; a memory unit having a set of digital position data, each representing a radial position of the scanning arm; a comparator that generates a plurality of clock pulses based, at least in part, on a comparison of the position signal with the position data; and an analog to digital converter that samples the emission signal when enabled by the plurality of clock pulses. The radial positions may be spaced in accordance with constant linear distances.
Additional preferred embodiments include a method for sampling an emission signal detected using a scanner having a scanning arm. The method includes providing an analog position signal representing a plurality of radial positions of the scanning arm; providing a set of digital position data, each representing a radial position of the scanning arm; generating a plurality of clock pulses based, at least in part, on a comparison of the position signal with the position data; and sampling the emission signal when enabled by the plurality of clock pulses. In another preferred embodiment, a scanning system includes a scanner and a computer. The scanner has one or more excitation sources that generate one or more excitation beams; an emission receiving element that receive an emission signal, responsive to the excitation beams, from a plurality of locations of a probe array; and a radial position generator that generate a plurality of radial positions of the emission receiving element. The computer includes a processor and a memory unit. Stored in the memory unit are a plurality of position data, each representing a radial position of the emission receiving element; and a set of comparator instructions that generate, in cooperation with the processor, a clock signal based, at least in part, on comparing one or more of the plurality of radial positions with one or more of the plurality of position data.
In accordance with another preferred embodiment, a computer program product described for use with a computer having a processor and a memory unit. The product includes a set of comparator instructions stored in the memory unit and executed in cooperation with the processor. The instructions generate a clock signal based, at least in part, on comparing one or more of a plurality of radial positions with one or more of a plurality of position data, wherein the plurality of radial positions are generated by a radial position generator of a scanning apparatus based on radial positions of an emission receiving element.
The above embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they be presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiment and implementations are illustrative rather than limiting.